B-1 cells constitute a unique set of B cells, with numerous distinguishing phenotypic and functional features. B-1 cells are an important object of study because they are responsible for the production of non-immune "natural" immunoglobulin, which provides protection against overwhelming infections prior to adaptive responses, and because B-1 cells have been linked to the pathological processes of malignancy and autoimmunity. Although B-1 cells appear first in ontogeny, their origin remains unclear. Evidence developed here indicates that B-1 cells in the spleen differ from B-1 cells in the peritoneal cavity in terms of a number of molecular characteristics. In conjunction with previous reports on various transgenic models, this has led to the hypothesis that splenic B-1 cells and peritoneal B-1 cells have separate origins-that the threshold required for B-1 cell differentiation following sIg engagement differs for B-1 cells derived from adult bone marrow, which preferentially populate the spleen, as compared to B-1 cells derived from fetal liver, which preferentially populate the peritoneal cavity. The broad, long term objective of this work is to understand the origin, features, and function of B-1 cells. This will be accomplished during the current project period through 3 specific aims. 1. Elucidate the origin of the transcriptional differences that distinguish B-1 populations, by identifying the molecular mechanisms responsible for regulating the biochemical state of PU.1, and by determining the consequences of such regulation; 2. Determine the molecular relationship between normal and immunoglobulin (Ig) transgenic splenic B-1 cells, normal and Ig transgenic peritoneal B-1 cells, tolerant Ig transgenic splenic B-2 cells, and normal splenic B-2 cells, in terms of transcription factor expression, gene expression and sIg signaling responses; and, 3. Test the hypothesis that alterations in signaling thresholds modulate development of splenic and peritoneal B-1 cells, and tolerant B-2 cells, using a series of radiation chimeras reconstituted with retrovirally transduced fetal liver or bone marrow stem cells, plus in vitro differentiation of B cells in fetal liver cultures following retroviral transduction, plus Ig transgenic mice. The information developed in this project will clarify the presently confused arena in which splenic and peritoneal B-1 cells are grouped together, and in so doing will set the stage for a more complete understanding of B-1 cells and will test the hypothesis that differences in signaling thresholds for B-1 cell precursors explain the contrasting results obtained to date from adoptive transfer and transgenic mouse experiments.